Metastasis is a major cause of mortality and remains a hurdle in the search for a cure for cancer. Not much is known about metastatic cancer cells and endothelial cross-talk, which occurs at multiple stages during metastasis. Here we report a dynamic regulation of the endothelium by cancer cells through the formation of nanoscale intercellular membrane bridges, which act as physical conduits for transfer of microRNAs. The communication between the tumour cell and the endothelium upregulates markers associated with pathological endothelium, which is reversed by pharmacological inhibition of these nanoscale conduits. These results lead us to define the notion of ‘metastatic hijack’: cancer cell-induced transformation of healthy endothelium into pathological endothelium via horizontal communication through the nanoscale conduits. Pharmacological perturbation of these nanoscale membrane bridges decreases metastatic foci in vivo. Targeting these nanoscale membrane bridges may potentially emerge as a new therapeutic opportunity in the management of metastatic cancer.

Intravasation and extravasation of cancer cells through blood/lymph vessel endothelium are essential steps during metastasis. Successful invasion requires coordinated tumor-endothelial crosstalk, utilizing mechanochemical signaling to direct cytoskeletal rearrangement for transmigration of cancer cells. However, mechanisms underlying physical interactions are difficult to observe due to the lack of experimental models easily combined with theoretical models that better elucidate these pathways. We have previously demonstrated that an engineered 3D in vitro endothelial-epithelial co-culture system can be used to isolate both molecular and physical tumor-endothelial interactions in a platform that is easily modeled, quantified, and probed for experimental investigation. Using this platform with mathematical modeling, we show that breast metastatic cells display unique behavior with the endothelium, exhibiting a 3.2-fold increase in interaction with the endothelium and a 61-fold increase in elongation compared to normal breast epithelial cells. Our mathematical model suggests energetic favorability for cellular deformation prior to breeching endothelial junctions, expending less energy as compared to undeformed cells, which is consistent with the observed phenotype. Finally, we show experimentally that pharmacological inhibition of the cytoskeleton can disrupt the elongatation and alignment of metastatic cells with endothelial tubes, reverting to a less invasive phenotype.

The future of cancer therapy relies on robust drug combinations. Traditional screening for drug discovery uses 2D monocultures. However, recent studies have shown that 3D cultures provide a more physiological platform to study cancer. Unfortunately, these systems do not capture interactions between tumor cells and the angiogenic component. The goal of this work is to establish a 3D in vitro co‐culture system combining these components to create a high‐throughput model to study cell interactions and screen anti‐angiogenic and anti‐tumorigenic therapies. Human endothelial cells were allowed to undergo tubulogenesis in a matrigel matrix to which metastatic breast cancer MDA‐MB‐231 cells were added. Interestingly, instead of forming embryoid bodies, the MDA cells align and incorporate into the endothelial vessels. Using this model system, we elucidate FAK and β1 integrin as mediators of the cell interactions. RNAi inhibition of FAK and β1 integrin resulted in the disruption of the architecture with 3.4 (p<0.001) and 8.6 (p<0.001) fold change in cell interactions, respectively. To probe downstream mediators of the FAK‐β1 signaling pathway, small molecule inhibitors were used to perturb this architecture, resulting in a 68% (p<0.05) decrease in invasive capacity. In conclusion, our studies show that a 3D co‐culture system provides a powerful platform for screening combination therapies.Supported by DoD BCRP.

Intercellular communication is a fundamental mechanism for maintaining cell homeostasis, facilitating the evolution of multicellular organisms. Classical modes of intercellular communication primarily focus on paracrine and endocrine signaling mechanisms or direct cellular exchange of cytoplasmic contents through gap-junctions. Recently, a novel mechanism of cellular communication was discovered that allows for direct transfer of intercellular contents through thin nanotubular projections forming continuous connections between cells. These structures, referred to as tunneling nanotubes (TNTs), have been found in a variety of eukaryotic and prokaryotic cell types. TNTs mediate transfer of a diverse array of intercellular contents including organelles, proteins, and pathogens. Here we report, TNTs allow for intercellular communication between metastatic breast cancer cells and the endothelium, introducing a potentially novel mechanism for breast cancer progression. Human endothelial cells were allowed to undergo tubulogenesis in a 3D matrigel matrix. To the preformed endothelial vessels, metastatic breast cancer MDA-MB-231 cells were added. The resulting phenotype was characterized using both fluorescent and electron microscopy. Interestingly, when added to the human endothelial cells, the metastatic breast cancer cells preferentially align and incorporate within the endothelial vessels, invading the tubular structures, instead of forming the expected mammary bodies. The physical proximity between the cell types facilitates the formation of TNTs that originate from the metastatic breast cancer cells to form continuous membrane connections with the endothelial vessels. To test for transfer of intercellular contents, the metastatic breast cancer cells were loaded with fluorescently labeled small molecules. Transfer of intercellular contents was quantified using both fluorescent microscopy and flow cytometry. The TNTs were found to transfer a variety of intercellular contents from the metastatic cells to the endothelium including green fluorescent protein, CFSE, and quantum dots. Between 20-40% of the isolated endothelial cell population received transfer of intercellular contents from the metastatic cells. Communication via the TNTs occurred quickly, with the membrane structures forming 4-6hrs post seeding, and intercellular transfer beginning at 6hrs and peaking at approximately 16-20hrs. In conclusion, TNTs provide a novel mechanism for communication between the tumor compartment and the angiogenic component, introducing a new paradigm of cancer progression where tumor cells can directly manipulate the surrounding cell populations to facilitate cancer pathology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4222. doi:1538-7445.AM2012-4222

Effective cancer therapy relies heavily on robust drug combinations. Our previous studies establish the advantage of 3D co‐culture model systems to study cancer‐promoting mechanisms. Here, we describe a model combining primary breast epithelial cells, HMEC, with a highly metastatic breast epithelial cell line, MDA‐MB‐231. We have identified a mode of communication between these cell types through thin cytoskeletal projections called tunneling nanotubes (TNTs). TNTs may promote cancer progression through rapid and specific transfer of cytoplasmic contents to neighboring cell populations resulting in transformation of previously healthy cells. TNTs form quickly (~1.5hrs) mediating transfer of intercellular contents to 35% (p<0.0001) of the primary cell population after 24hr co‐culture. The TNTs are composed of cytoskeletal components and are inhibited by low dose actin and tubulin small molecule inhibitors. Combination of 500pM Docetaxel with 30nM Latrunculin A or 50nM Cytoclasin D resulted in a 3× (p< 0.01) disruption of TNT structures, reducing TNT‐mediated communication by 33.14% (p<0.001) and 61.96% (p<0.0001), respectively. In conclusion, TNTs present a novel mechanism for breast cancer progression, inhibited by actin‐tubulin small molecule inhibitors. Therefore, low dose dual combination actin tubulin inhibitors are strong candidates for breast cancer therapies. Supported by DoD BCRP.